The discoidin domain receptors DDR and DDR are
The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases (RTKs) that are stimulated by collagen in the extracellular matrix (ECM). Unlike most other RTKs, they form ligand-independent stable dimers that are non-covalently linked. This ECM activation induces receptor phosphorylation with slow kinetics, i.e., a slow and prolonged response, unlike the quicker responses of most other RTKs. DDRs regulate cell proliferation, differentiation, migration, and survival and control extracellular matrix homeostasis and remodeling. Dysregulated DDR function has been associated with fibrosis, arthritis, and cancer.3, 4DDR2 is predominantly expressed in fibroblasts, chondrocytes, osteoblasts, and other connective-tissue cells of mesenchymal origin. Both the p.Leu610Pro and the p.Tyr740Cys substitutions are located in the kinase domain of the DDR2 receptor. To study the consequences of these substitutions, we first examined DDR2 autophosphorylation. All results were reproduced in at least two independent experiments. Control fibroblasts and fibroblasts from individuals 1 and 3, heterozygous for the p.Leu610Pro and the p.Tyr740Cys substitutions, respectively, were cultured in Dulbecco's modified Eagle's medium (DMEM)—high glucose (Lonza) supplemented with 10% fetal calf serum, penicillin, streptomycin, and glutamine. When the cells were 80%–90% confluent, fresh medium was added, and the cells were harvested the following day. The total phosphorylated DDR2 was measured with a DuoSet IC Phospho-DDR2 kit (#DYC6170, R&D Systems) according to the manufacturer’s recommendations (see Supplemental Methods). More phosphorylated DDR2 was observed in the fibroblasts from affected individuals than in those of controls, indicating that the variants were activating and caused autophosphorylation of the receptor (Figures 4A and 4B). DDR2 has 14 tyrosine residues: four located in the extracellular juxtamembrane region, and the rest located in the kinase domain of the receptor. Tyr740 is thought to play a critical role for DDR2 autoinhibition and site-directed Tyr740Phe mutagenesis caused in vitro DDR2 autophosphorylation and thereby mimicked the effect of high levels of SRC. Because the p.Tyr740Cys variant alters this tyrosine residue, phosphorylation at this site was further examined. For immunoblot analyses, cells were starved of serum overnight before being harvested, separated on a high-resolution gel system, transferred to nitrocellulose membranes, and incubated overnight at 4°C with MG-132 against phospho-Tyr740-DDR2 (#MAB25382) and DDR2 (#MAB2538) (R&D Systems, detailed description in Supplemental Methods). Anti-rabbit IgG (#7074) and anti-mouse IgG (#7076) (Cell Signaling Technology) were used as secondary antibodies. As a control for equal loading, the membranes were blocked again, incubated overnight with a GAPDH primary antibody (#G99545, Sigma-Aldrich), and visualized as described above. HEK293 cells transiently transfected with a human DDR2 expression vector were used as a positive control (see Supplemental Methods). After use of an antibody against non-phosphorylated DDR2, no DDR2 was detected in fibroblasts from affected individuals or controls (Figure S5). In contrast, the antibody against Tyr740-phosphorylated DDR2 left a clear band of expected size in fibroblasts harboring the p.Leu610Pro and p.Tyr740Cys substitutions, but not in controls (Figure 4B and Figure S6). Increased phosphorylation of Tyr740 in cells heterozygous for the p.Tyr740Cys variant suggested that autophosphorylation of the wild-type protein partner of a DDR2 dimer took place in these cells or that this antibody also binds to DDR2 if phosphorylated at nearby tyrosine residues, e.g., Tyr736 and Tyr741 (Figure 3). It is likely that both variants cause ligand-independent kinase activation, as has been described for other RTKs. The DDRs can interact with multiple proteins and also modulate signaling pathways initiated by other matrix receptors, cytokines,growth factors, and transmembrane receptors in a context- and cell-type-dependent manner.3, 4 We therefore evaluated the consequences of DDR2 activation on potential downstream growth-stimulatory pathways and STAT1. The latter is an important modifier in the overgrowth and tissue wasting seen in individuals with PDGFRB [MIM: 173410] gain-of-function variants. The following proteins were assessed with appropriate antibodies, all obtained from Cell Signaling Technology at recommended dilutions (see Supplemental Methods for details): phospho-Tyr542-PTPN11(SHP-2), phospho-Tyr580-PTPN11(SHP-2), PTPN11(SHP-2), phospho-Ser473-AKT, AKT, phospho-Thr202/Tyr204-MAPK3(ERK1), MAPK3(ERK1), phospho-Tyr416-SRC, Tyr416-SRC, phospho-Tyr527-SRC, Tyr527-SRC, SRC, and phospho-Tyr70-STAT1. We did not detect increased phosphorylation of any of these proteins (Figures S7–S21). This suggests that the consequence of DDR2 activation in Warburg-Cinotti syndrome (MIM: 618175) is targeted to a group of proteins with little signal-transduction crosstalk with well-known growth-stimulatory pathways, such as the RAS/ERK and PI3K/AKT pathways.